ATN-224 (choline tetrathiomolybdate) is a copper-binding compound that primarily targets and inhibits superoxide dismutase 1 (SOD1). It inhibits purified bovine SOD1 with an IC50 of 0.33 ± 0.03 μmol/L after 16 hours of incubation. [1]
It also inhibits intracellular SOD1 activity in human umbilical vein endothelial cells (HUVEC) with an IC50 of 0.0175 ± 0.0037 μmol/L. [1]
The compound has a high and specific affinity for copper ions (Kd ~10⁸ mol/L⁻¹) and shows no binding to calcium, iron, magnesium, zinc, or manganese ions at concentrations up to 1 mmol/L. [1]
It is a poor inhibitor of cytochrome c oxidase, with significant inhibition (50%) observed only at 1 mmol/L in isolated mitochondrial preparations. [1]

According to isothermal titration calorimetry, ATN-224 has a high specific affinity for copper ions (108 mol/L-1) and does not bind calcium, iron, magnesium, zinc, or manganese ions at concentrations up to 1 mM. Both HUVECs' proliferation was decreased by ATN-224 (IC50=1.4±0.3 μM; n=5). Purified bovine SOD1 activity is also inhibited by ATN-224, with an IC50 of 0.33±0.03 μM during a 24-hour incubation period. The maximal inhibition of SOD1 by ATN-224 is reached after roughly 16 hours, and this effect is time-dependent. It seems that ATN-224 inhibits SOD1 by reducing copper enzyme levels. With an IC50 of 17.5±3.7 nM, ATN-224 can dose-dependently reduce SOD1 activity in endothelial cells. ATN-224 suppresses ERK1/2 phosphorylation produced by FGF-2 in a time- and dose-dependent manner; its IC50 ranges from 1.25 to 2.5 μM, which is in line with the IC50 for preventing proliferation [1]. ATN-224 is a tiny inorganic compound that can be taken orally that suppresses the activity of the copper/zinc-dependent enzyme superoxide dismutase 1 (Cu/Zn-SOD1) in tumor cells and endothelial cells [2].

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ATN-224 inhibits the proliferation of HUVEC with an IC50 of 1.4 ± 0.3 μmol/L, but has little effect on confluent, quiescent HUVEC. It also inhibits human microvessel endothelial cell proliferation. [1]
ATN-224 inhibits the proliferation of the multiple myeloma cell line MM1S with an IC50 of approximately 6.5 μmol/L. [1]
It inhibits SOD1 activity in MM1S cells with an IC50 of approximately 0.04 μmol/L. [1]
In HUVEC, ATN-224 treatment (1 μmol/L for 72 hours) leads to an increase in steady-state levels of superoxide anions, as measured by dihydroethidine staining. [1]
ATN-224 inhibits FGF-2- and VEGF-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in HUVEC in a dose- and time-dependent manner, with complete inhibition observed after 48 hours of preincubation with 10 μmol/L. [1]
In contrast to endothelial cells, ATN-224 (10 μmol/L for 48 hours) induces apoptosis in MM1S multiple myeloma cells, as detected by increased cleaved poly(ADP-ribose) polymerase (PARP) and activated caspase-3. [1]
The effects of ATN-224 on HUVEC proliferation, angiogenesis, and ERK phosphorylation, as well as on tumor cell apoptosis, could be substantially reversed by the cell-permeable SOD mimetic Mn(III) tetrakis(4-benzoic acid) porphyrin chloride (Mn-TBAP) or by preloading cells with polyethylene glycol-conjugated SOD (PEG-SOD). [1]
Preloading ATN-224 with copper ions in the presence of albumin abrogated its antiproliferative and SOD1 inhibitory activities in HUVEC. [1]
ATN-224 is taken up by HUVEC in a dose-dependent manner and is retained for at least 24 hours after removal from the culture medium. The intracellular concentration of ATN-224 per HUVEC was calculated to be ~411 μmol/L at the highest concentration tested (20 μmol/L in medium). [1]

In the mouse Matrigel plug model, ATN-224 also strongly (P<0.05) reduced angiogenesis when it was included into the plugs or given orally by gavage. Inhibition of angiogenesis happens prior to detectable copper depletion in the plasma or from the Matrigel plug when ATN-224 is given by oral gavage. This finding suggests that ATN-224 suppresses angiogenesis without requiring copper to be consumed [1].

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In the Matrigel plug model of angiogenesis in female BALB/c nude mice, ATN-224 administered by oral gavage (daily, Monday to Friday) significantly inhibited growth factor-driven angiogenesis. This inhibition occurred before any measurable depletion of copper in plasma or in the Matrigel plug. [1]
The antiangiogenic effect of ATN-224 in the Matrigel plug could be reversed by co-administration of the SOD mimetic Mn-TBAP directly into the plug. [1]

Cytochrome c Oxidase Activity Assay: The effect of ATN-224 on cytochrome c oxidase was measured using isolated mouse liver mitochondria. Mitochondria were incubated with various concentrations of the compound for 16 hours at 4°C, and enzyme activity was assessed using a commercial assay kit. [1]
Purified SOD1 Activity Assay: The inhibitory effect of ATN-224 on purified bovine SOD1 was measured using a water-soluble tetrazolium salt (WST-1) assay. Superoxide anions generated by xanthine oxidase reduce the WST-1 to a formazan dye. SOD1 inhibits this reduction. The compound was incubated with SOD1 for various times (up to 16 hours) before the activity was measured. A standard curve was generated using bovine SOD. [1]
Intracellular SOD1 Activity Assay: HUVEC or MM1S cells were treated with ATN-224 for 16 hours, then lysed. SOD1 activity in the lysates was assayed using the same WST-1-based method, measuring the inhibition of superoxide-mediated tetrazolium salt reduction. [1]
Copper Depletion from SOD1: ATN-224 was incubated with purified SOD1 protein for 30 minutes. The protein was then repurified by gel filtration chromatography, and its copper content was determined using inductively coupled plasma-mass spectrometry (ICP-MS). [1]

Cell Proliferation Assay: For HUVEC, cells were plated on gelatin, stimulated with 2 ng/mL FGF-2, and treated with ATN-224 for 48 hours. Proliferation was determined using Alamar Blue or MTT assays. For MM1S multiple myeloma cells, cells were plated in T-75 flasks and incubated with the compound for 48-96 hours. Proliferation was determined using calcein AM staining. [1]
Western Blot Analysis: For ERK phosphorylation studies, HUVEC were preincubated with ATN-224 for various times and doses, then stimulated with 10 ng/mL FGF-2 or VEGF for a short period. Cell lysates were analyzed by Western blot using an antibody specific for phosphorylated ERK1/2 (Thr202/Tyr204). Total ERK1/2 antibody was used for signal correction. For apoptosis studies, MM1S cells were treated with ATN-224 for 48 hours, and cytoplasmic/nuclear fractions were analyzed by Western blot for cleaved PARP and activated caspase-3. [1]
Superoxide Anion Detection (Dihydroethidine Staining): HUVEC were treated with 1 μmol/L ATN-224 for 72 hours. Cells were then incubated with 5 μmol/L dihydroethidine (for microscopy) or 1.5 μmol/L (for flow cytometry). The oxidized product of dihydroethidine yields red fluorescence, which was visualized by microscopy or quantified by flow cytometry. [1]
Subcellular Fractionation: Cytoplasmic and nuclear fractions from cells were prepared by a standard differential centrifugation method. Mitochondria were freshly isolated from mouse livers using a commercial kit. [1]

Matrigel Plug Angiogenesis Model:** Female BALB/c nude mice (4-8 weeks old) were used. Cold Matrigel was mixed with 800 ng/mL FGF-2 or 300 ng/mL VEGF and 50 μg/mL heparin. The mixture was injected subcutaneously. ATN-224 was administered either by oral gavage (daily, Monday to Friday) or added directly to the Matrigel plug (94 μmol/L). In some experiments, the SOD mimetic Mn-TBAP (100 μmol/L) was also added directly to the plug. After 5 days, the plugs were excised, homogenized, and hemoglobin content was measured using Drabkin's solution to quantify angiogenesis. [1]

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Matrigel Plug Angiogenesis Model: Female BALB/c nude mice (4-8 weeks old) were used. Cold Matrigel was mixed with 800 ng/mL FGF-2 or 300 ng/mL VEGF and 50 μg/mL heparin. The mixture was injected subcutaneously. ATN-224 was administered either by oral gavage (daily, Monday to Friday) or added directly to the Matrigel plug (94 μmol/L). In some experiments, the SOD mimetic Mn-TBAP (100 μmol/L) was also added directly to the plug. After 5 days, the plugs were excised, homogenized, and hemoglobin content was measured using Drabkin's solution to quantify angiogenesis. [1]

ATN-224 is taken up by cells in a dose-dependent manner and is retained intracellularly for at least 24 hours after removal from the culture medium. [1]
The compound is administered orally (by gavage) in vivo. [1]
The inhibition of angiogenesis in the Matrigel plug model occurred before any measurable depletion of copper in either plasma or the Matrigel plug itself, indicating that its antiangiogenic effect is independent of systemic copper depletion. [1]

ATN-224 was well tolerated. The most frequent non-hematological adverse events were fatigue, gastrointestinal disorders, and skin reactions. Hematological toxicities included reversible grade 3-4 leucopenia. [2]
Three patients in the high-dose arm discontinued the study drug due to grade 3-4 toxicities: one due to leucopenia, one due to generalized rash, and one due to elevated aminotransferases. [2]
For patients with mild-to-moderate (grade 1-2) adverse events related to ATN-224, symptomatic treatment was administered and therapy continued. For severe or life-threatening (grade 3-4) adverse events that could be treated symptomatically to grade 2 or less, appropriate therapy was given without dose delay or adjustment. Dose reductions were made for persistent grade 3-4 toxicities (>14 days). [2]

[1]. Copper binding by tetrathiomolybdate attenuates angiogenesis and tumor cell proliferation through the inhibition of superoxide dismutase 1. Clin Cancer Res. 2006 Aug 15;12(16):4974-82.

[2]. A non-comparative randomized phase II study of 2 doses of ATN-224, a copper/zinc superoxide dismutase inhibitor, in patients with biochemically recurrent hormone-na?ve prostate cancer. Urol Oncol. 2013 Jul;31(5):581-8.

Tiomolibdate acid choline is an orally effective second-generation tetrathiomolybdate analog with anti-angiogenic and antitumor activities. Tiomolibdate acid choline selectively chelates copper ions in superoxide dismutase 1 (SOD1) in endothelial cells, thereby depleting copper in SOD1 and inhibiting its activity. Inhibition of SOD1 interferes with the activation of multiple signal transduction pathways required for cell proliferation and angiogenesis, including ERK1/2, FAK, and Src kinase-mediated pathways. This leads to inhibition of cell proliferation and angiogenesis and induces apoptosis.

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Drug Indications
Treatment of Wilson's disease

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ATN-224 (choline tetrathiomolybdate) is a second-generation, orally available copper-binding compound derived from ammonium tetrathiomolybdate. It is being developed for the treatment of cancer and was in two phase I clinical trials for advanced solid and hematologic malignancies at the time of publication. This study provides the first validation of SOD1 as a therapeutic target for the inhibition of angiogenesis and tumor growth, identifying the specific molecular target of ATN-224. The compound appears to have distinct effects on different cell types: it inhibits proliferation in endothelial cells without inducing apoptosis, while it induces apoptosis in tumor cells like multiple myeloma. This differential activity may provide a synergistic antitumor effect by targeting both the tumor and its vasculature. [1]

C10H28MON2O2S4

434.5574

436.024

649749-10-0

18442052

Pink to red solid powder

4

6

4

19

65.7

0

[Mo](=S)=S.[S-][H].[S-][H].O([H])C([H])([H])C([H])([H])[N+](C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H].O([H])C([H])([H])C([H])([H])[N+](C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

NEYVHGQOGHJAAD-UHFFFAOYSA-N

InChI=1S/2C5H14NO.Mo.4S/c2*1-6(2,3)4-5-7/h2*7H,4-5H2,1-3H3/q2*+12*-1

2-hydroxy-N,N,N-trimethylethan-1-aminium tetrathiomolybdate

Bis-choline tetrathiomolybdate Decuprate ATN-224 ATN 224 ATN224

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~115.59 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.78 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.78 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)